The long-term, overall objective of this project is to elucidate the mechanisms underlying the secretion of saliva and of its two major fractions, the water/electrolyte and macromolecular fractions. The more immediate goal of the studies described in this proposal is to characterize the ion transport systems which are involved in the formation of the fluid/electrolyte fraction of primary or precursor saliva by acinar cells. The proposed studies are based on previous evidence indicating that salivary fluid originates primarily in these cells and that its secretion is inhibited by ouabain and by furosemide. A model of the ionic mechanism of salivary fluid secretion is proposed, involving, as a first step, a Na-coupled, furosemide-sensitive Cl-entry into the cells, which is driven by the Na gradient generated by an ouabain-sensitive Na, K pump. We propose to use, therefore, dispersed acini isolated from the rat submandibular gland to investigate the presence and activation of these two transport systems. The specific aims are to measure: 1) furosemide-sensitive fluxes of Na-22 and Cl-36. Both isotope exchange and net fluxes will be measured before and after exposure to cholinergic and adrenergic agents. The effects of ouabain, of changes in the ionic composition (Na, C1, K, Ca) of the incubation medium, of the Ca++ ionophore A23187, of exogenous derivatives of cAMP and cGMP and of substances which alter cell nucleotide content (such as forskolin and Na nitroprusside) will also be investigated. 2) The rate of binding of H-3 ouabain. The time required for half maximal and maximal binding, association, dissociation and equilibrium dissociation (KD) constants and density of binding sites (Bmax) will be measured under the same experimental conditions indicated for isotope fluxes. 3) Ouabain-sensitive Rb-86 uptake. Rates of uptake will be measured in the same experimental conditions as H-3-ouabain binding. The results of these studies should provide more direct evidence for the presence and activation of two ion transport systems involved in the secretion of fluid and electrolytes by salivary acinar cells and contribute to our understanding of the mechanisms of saliva formation.